Manganese-dependent peroxidases (or manganese peroxidases) are ligninolytic enzymes produced by white rot fungi. One such fungus is the basidiomycete Phanerochaete chrysosporium which is capable of degrading lignin to the point of mineralization with H2O and CO2 as the final products. This degrading ability is due to the exocellular peroxidases such as various isozymes of lignin peroxidases (LiP) and manganese peroxidases (MnP) along with an extracellular H2O2-generating system. Manganese peroxidases are glycosylated heme protein peroxidases that catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. Mn3+ is subsequently chelated by organic acids to create the diffusible oxidants that attack phenolic lignin structures (Kishi et al. 1994. Mechanism of manganese peroxidase compound II reduction. Effect of organic chelators and pH. Biochemistry 33: 8694–8701).
Several isozymes of manganese peroxidases from the fungus Phanerochaete chrysosporium have been described (Tien and Kirk. 1983. Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds. Science 221: 661–663; Glenn et al. 1983. An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114: 1077–1083; Kuwahara et al. 1984. Separation and characterization of two extracellular H2O2-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett. 169: 247–250) and the major isozyme, MnPI (H3) has been characterized in detail (Gold and Alic. 1993. Molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Microbiol. Rev. 57:605–622) and its X-ray structure reported (Sundaramoorthy, M., K. Kishi, M. H. Gold, and T. L. Poulos. 1994. The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution. J Biol. Chem. 269:32759–32767). These isozymes are encoded by a family of structurally related genes that are expressed under nutrient-limiting conditions during secondary metabolic growth phase of the fungus (Gettemy et al. 1998. Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family. Appl Environ Microbiol 64(2): 569–74).
Commercial production of MnP enzymes has its application in the fields of paper making, waste treatment, bioremediation and others. In the pulp and paper industry, biological pulping and biological bleaching have the potential of improving the quality of pulp and paper, reducing energy costs and environmental pollution relative to traditional pulping and bleaching operations (U.S. Pat. No. 5,691,193). The technology has focused on white rot fungi that have complex extracellular ligninolytic enzymes such as MnP and LiP. Unlike the xylanases used in commercial bleaching to degrade hemicelluloses, peroxidases such as LiP and MnP have not been much tested in applications for manufacturing processes. This is simply due to the fact that effective methods for the production of commercially viable yields of enzyme have not been developed. Scale-up to industrial process requirements presents challenges that are difficult to simulate in the laboratory or pilot-scale tests. Thus there is a need in industry for large-scale production of ligninolytic enzymes such as MnP.
Large-scale production of MnP may also be employed in the treatment of environmental pollutants such as the cleanup of textile mill effluents as well as the bioremediation of dye-contaminated soil. For example, textile effluents cause a high environmental impact when released into the environment without correct treatment. Azo dyes are important synthetic compounds that are widely used in the dyestuff and textile industries. They are not biodegradable and tend to persist in the environment unless subjected to costly physical-chemical decontamination processes. Disperse Yellow 3 [2-(4′-acetamidophenylazo)-4-methylphenol] (DY3) which is an important yellow azo dye used in the industry, is a carcinogen. It was reported that the degradation of DY3 to CO2 is possible by MnP (Spadaro and Renganathan. 1994. Peroxidase-catalyzed oxidation of azo dyes: mechanism of disperse Yellow 3 degradation. Arch Biochem Biophys. 312 (1): 301–307). Another example of environmental pollutants is the class of compounds called chlorophenols. 2,4,6,-Trichlorophenol and pentachlorophenol have been extensively used as wood preservatives and pesticides (Freiter. 1979. Chlorophenols, p 864–872. In “Encylopedia of chemical technology”. Mark, Othmer, Overberger and Seaborg (eds). Vol 5. John Wiley & Sons, Inc. New York, N.Y. and Rappe. 1980. Chloroaromatic compounds containing oxygen, phenols, diphenyl ethers, dibenzo-p-dioxins, and dibenzofuran, p 157–179. In Hutzinger (ed), The handbook for environmental chemistry. Springer-Verlag K G, Berlin, Germany). In addition, 2,4-dichlorophenol and 2,4,5-trichlorophenol are precursors in the synthesis of herbicides (Freiter, supra). It has been reported that the degradation of such persistant environmental pollutants by Phanerochaete chrysosporium involves an initial dechlorination step catalyzed by either LiP or MnP (Reddy et al. 1998. Degradation of 2,4,6-Trichlorophenol by Phanerochaete chrysosporium: Involvement of Reductive Dechlorination; Joshi and Gold. 1993. Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59:1779–1785; and Valli and Gold. 1991. Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J. Bacteriol. 173:345–352).
MnP can also be employed for bioremediation. For example, U.S. Pat. No. 6,268,204 describes how MnP is used to remediate liquid or solid waste streams containing organo-halides.
Fungal MnP is also capable of degrading aminonitrotoluenes, the main intermediates of the explosive 2,4,-trinitrotoluene (TNT). Radioactive experiments using a complex mixture of uniform ring-labeled 14C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase (Scheibner and Hofrichter. 1998. Conversion of aminonitrotoluenes by fungal manganese peroxidase. J Basic Microbiol. 38(1): 51–59).
MnP can also be employed in the synthesis of phenolic and aromatic amine polymers such as poly(p-ehylphenol) and poly(m-cresol) to help control product yields, molecular weight, molecular weight distribution and polydispersity (U.S. Pat. No. 6,096,859). Such polymers are important constituents of coatings, laminates and intergrated circuit chips. U.S. Pat. No. 5,608,040 reports a process for producing lignin-containing polymers in the presence of radical oxidizing enzymes such as MnP.
Several endeavors for over-expressing MnP in a variety of hosts have been reported. Attempts to express MnP genes in bacteria have resulted in the production of inclusion bodies containing catalytically inactive enzyme. The reason is that prokaryotic organisms such as bacteria inherently lack the ability to synthesize heme, a necessary component of the native enzyme (Andrawis et al. (eds) 1990. Biotechnology in Pulp and Paper Manufacture; Applications and Fundamental Investigations. Butterworth-Heinemann, Butterworth-Heinemann, Boston, 601). Efforts to optimize this system involved refolding of the inactive polypeptides into active enzyme under specific conditions (2 M urea, pH 8.0, in the presence of CaCl2, hemin, and oxidized glutathione) (Whitwam and Tien. 1996. Heterologous expression and reconstitution of fungal Mn peroxidase. Arch Biochem Biophys 15; 333(2):439–46). Still, however, yields were low for large-scale commercial production.
Another system reported for MnP expression is the baculovirus expression system (Pease et al. 1991. Heterologous expression of active manganese peroxidase from Phanerochaete chrysosporium using the baculovirus expression system. Biochem. Biophys. Res. Commun. 179:897–903). This system is capable of producing biochemically active enzyme, indicating proper post-translational modifications, and enzymatic activity could be further enhanced (up to 15-fold increase) upon the addition of hemin at 1 ug/ml to the medium. Still, however, yields are not appreciably higher than those observed in P. chrysosporium cultures. The system also suffers from a serious limitation, its' high production costs. The addition of hemin to the medium is not a cost-effective measure since one gram of hemin crystals can cost about $27.
Expressing MnP in fungal systems also has its pitfalls. Attempts included the expression of exogenous sequences (heterologous expression) as well as the overexpression of endogenous MnP genes (homologous expression). Homologous expression of a recombinant MnP gene was attempted in Phanerochaete chrysosporium. An endogenous MnP gene was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Recombinant MnP yields were higher than those of the baculovirus expression systems. Heme insertion, folding, and secretion were normal (Mayfield et al. 1994. Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium. Appl Environ Microbiol. 60(12): 4303–9). Although the expression levels in this system were good enough for structural and functional studies of recombinant MnP, they were not high enough to support cost-effective large-scale industrial production.
Heterologous expression of recombinant MnP in the commercially available fungal systems, Aspergillus oryzae and Aspergillus niger, has also been attempted and shown to be possible. However, success was limited by poor yield and by the small fraction of peroxidase polypeptide that is assembled into a functional enzyme. In the Aspergillus oryzae system, MnP expression was attempted using a vector in which the cDNA of mnp-1 from Phanerochaete chrysosporium was fused with the A. oryzae Taka amylase promoter and secretion signal. Yields of 5 mg rMnP/L were obtained and optimal expression required 500 mg/L hemin in the medium. Lowered concentrations of hemin resulted in decreased yields (Stewart et al. 1996. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae. Appl. Environ. Microbiol. 62:860–864). In the Aspergillus niger system, overexpression of MnP was unsuccessful. Problems with this system included low specific activity of the recombinant MnP protein, low yields and degradation of a recombinant MnP: GLA fusion protein. The specific activity of rMnP was lower than that of the native enzyme. The initial yields obtained for rMnP in A. niger MGG029 were 5 to 10 mg/liter, which is low compared with other fungal proteins expressed in filamentous fungi. These yields could be increased to 100 mg of extracellular rMnP/liter under hemoglobin supplementation conditions (Conesa et al. 2000. Studies on the production of fungal peroxidases in Aspergillus niger. Appl Environ. Microbiol. 66(7): 3016–23).
The failure of genetically engineered bacterial or fungal systems to produce commercially high levels of MnP has prompted scientists to investigate the plant system. Transgenic plants offer the potential to be one of the most economical systems for large-scale production of proteins for industrial, pharmaceutical, veterinary and agricultural use. Advantages of plant systems include the low cost of growing a large biomass, easy scale-up (increase of planted acreage), natural storage organs (tubers, seeds), and established practices for efficient harvesting, transporting, storing and processing of the plant. Recombinant proteins can be targeted to seeds allowing stable storage of the recombinant proteins for extended periods. Plants offer advantages over other production systems since some proteins may be used without extensive purification, because for many applications, plant material is used directly as a food source or feed stock.
The only example attempting the expression of MnP in plants has failed. Austin et al. report that expressing the P. chrysosporium mnp-1 gene in alfalfa had deleterious effects on plant growth and development in vitro or in the greenhouse. Transgenic alfalfa plants were stunted and flowered later than control plants and the highest-expressing plants, with MnP levels above 0.3% soluble protein, died (Austin et al. 1995. Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase. Euphytica 85: 381–93).
The inventors have discovered that it is possible to obtain high level expression of a recombinant, fungally-derived nucleic acid sequence encoding MnP in corn seeds. It is, therefore, an object of the present invention to produce amounts of recombinant MnP (rMnP) that by far exceed the current capacity of traditional recombinant protein sources such as filamentous fungi or bacteria.
A further object of the invention is to produce MnP in plants such that the plant is viable, that is, lives to provide a source of MnP and does not die.
Another object of the invention is the application of large-scale production of MnP to industrial markets for which it had previously been economically unfeasible to enter.
Yet another object of the present invention is to produce rMnP in quantities large enough as to provide considerable cost savings for the industries.
An object of this invention is the development of a plant expression system that allows the efficient and large-scale production of heterologous proteins.
Another object of the invention is to preferentially express the rMnP in the seed of the plant.
A still further object of the invention is to direct expression of rMnP in plants to the cell wall of the plant.
An object of the invention is to further improve expression of rMnP in plants by backcrossing transgenic plants containing the MnP expressing gene into plants with good agronomic traits.
The objectives of this invention will become apparent in the description below. All references cited are incorporated herein by reference.